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Functional and Structural Characterization of RsbU, a Stress Signaling Protein Phosphatase 2C

2004; Elsevier BV; Volume: 279; Issue: 39 Linguagem: Inglês

10.1074/jbc.m405464200

1083-351X

Olivier Delumeau, Sujit Dutta, Matthias Brigulla, Grit Kuhnke, Steven W. Hardwick, Uwe Völker, Michael D. Yudkin, Richard J. Lewis,

ATP Synthase and ATPases Research

RsbU is a positive regulator of the activity of σB, the general stress-response σ factor of Gram+ microorganisms. The N-terminal domain of this protein has no significant sequence homology with proteins of known function, whereas the C-terminal domain is similar to the catalytic domains of PP2C-type phosphatases. The phosphatase activity of RsbU is stimulated greatly during the response to stress by associating with a kinase, RsbT. This association leads to the induction of σB activity. Here we present data on the activation process and demonstrate in vivo that truncations in the N-terminal region of RsbU are deleterious for the activation of RsbU. This conclusion is supported by comparisons of the phosphatase activities of full-length and a truncated form of RsbU in vitro. Our determination of the crystal structure of the N-terminal domain of RsbU from Bacillus subtilis reveals structural similarities to the regulatory domains from ubiquitous protein phosphatases and a conserved domain of σ-factors, illuminating the activation processes of phosphatases and the evolution of "partner switching." Finally, the molecular basis of kinase recruitment by the RsbU phosphatase is discussed by comparing RsbU sequences from bacteria that either possess or lack RsbT. RsbU is a positive regulator of the activity of σB, the general stress-response σ factor of Gram+ microorganisms. The N-terminal domain of this protein has no significant sequence homology with proteins of known function, whereas the C-terminal domain is similar to the catalytic domains of PP2C-type phosphatases. The phosphatase activity of RsbU is stimulated greatly during the response to stress by associating with a kinase, RsbT. This association leads to the induction of σB activity. Here we present data on the activation process and demonstrate in vivo that truncations in the N-terminal region of RsbU are deleterious for the activation of RsbU. This conclusion is supported by comparisons of the phosphatase activities of full-length and a truncated form of RsbU in vitro. Our determination of the crystal structure of the N-terminal domain of RsbU from Bacillus subtilis reveals structural similarities to the regulatory domains from ubiquitous protein phosphatases and a conserved domain of σ-factors, illuminating the activation processes of phosphatases and the evolution of "partner switching." Finally, the molecular basis of kinase recruitment by the RsbU phosphatase is discussed by comparing RsbU sequences from bacteria that either possess or lack RsbT. Reversible phosphorylation of proteins is the predominant regulatory mechanism in biology, modulating cellular processes such as signaling, division, and development. The phosphorylation of regulatory proteins by protein kinases effects a change in their function and structure (1Johnson L.N. Lewis R.J. Chem. Rev. 2001; 101: 2209-2242Crossref PubMed Scopus (506) Google Scholar), reversed by the action of protein phosphatases, which restore the regulatory proteins to their original, unphosphorylated state. Hence the cellular response is determined by controlling the enzymatic activities of the mutually antagonistic kinases and phosphatases. Protein phosphatases can be divided into three major groups, defined by their substrate specificity (2Kennelly P.J. Chem. Rev. 2001; 101: 2291-2312Crossref PubMed Scopus (65) Google Scholar): phosphotyrosine (further subdivided into Cdc25 and the conventional and the low molecular weight phosphotyrosine phosphatases), phosphoserine/threonine (further subdivided into protein phosphatase P and M families), and phosphoaspartate phosphatases (e.g. Rap and Spo0E from Bacillus). In addition, there is the dual-specific phosphatase group, which can dephosphorylate phosphotyrosine and phosphoserine/threonine substrates, and it has been recorded that several histidine kinases also have phosphohistidine phosphatase activity (3Zhu Y. Qin L. Yoshida T. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7808-7813Crossref PubMed Scopus (102) Google Scholar).In Bacillus subtilis, five serine/threonine protein phosphatases have been identified that belong to the PP2C subgroup of the protein phosphatase M family, namely PrpC (4Obuchowski M. Madec E. Delattre D. Boel G. Iwanicki A. Foulger D. Seror S.J. J. Bacteriol. 2000; 182: 5634-5638Crossref PubMed Scopus (51) Google Scholar), SpoIIE (5Duncan L. Alper S. Arigoni F. Losick R. Stragier P. Science. 1995; 270: 641-644Crossref PubMed Scopus (196) Google Scholar), RsbP (6Vijay K. Brody M.S. Fredlund E. Price C.W. Mol. Microbiol. 2000; 35: 180-188Crossref PubMed Scopus (154) Google Scholar), RsbX and RsbU (7Yang X. Kang C.M. Brody M.S. Price C.W. Genes Dev. 1996; 10: 2265-2275Crossref PubMed Scopus (236) Google Scholar). PrpC plays an important regulatory role in stationary phase (8Gaidenko T.A. Kim T.J. Price C.W. J. Bacteriol. 2002; 184: 6109-6114Crossref PubMed Scopus (69) Google Scholar), and SpoIIE regulates differentiation in Bacillus, a process known as sporulation, by forming a complex with the cell division protein FtsZ (5Duncan L. Alper S. Arigoni F. Losick R. Stragier P. Science. 1995; 270: 641-644Crossref PubMed Scopus (196) Google Scholar, 9Lucet I. Feucht A. Yudkin M.D. Errington J. EMBO J. 2000; 19: 1467-1475Crossref PubMed Scopus (62) Google Scholar). RsbX, RsbP, and RsbU are involved in the regulation of the alternative σ factor, σB, which controls the general stress response of B. subtilis and other Gram+ microorganisms, such as the human pathogens Listeria monocytogenes and Staphylococcus aureus (10Hecker M. Völker U. Adv. Microb. Physiol. 2001; 44: 35-91Crossref PubMed Scopus (252) Google Scholar, 11Price C.W. Sonenshein A.L. Hoch J.A. Losick R. Bacillus subtilis and Its Closest Relatives: from Genes to Cells. ASM Press, Washington, D. C.2002: 369-384Google Scholar). Depending on the nature of the stress, the signal is conveyed to σB by two separate pathways, which converge on phosphorylated RsbV (RsbV-P), 1The abbreviations used are: RsbV-P, phosphorylated RsbV; IPTG, isopropyl-1-thio-β-d-galactopyranoside; TPR, tetratricopeptide repeat; PP5, protein phosphatase 5. 1The abbreviations used are: RsbV-P, phosphorylated RsbV; IPTG, isopropyl-1-thio-β-d-galactopyranoside; TPR, tetratricopeptide repeat; PP5, protein phosphatase 5. the common substrate for RsbP and RsbU (Fig. 1). A decrease in the intracellular energy level activates RsbP, via RsbQ (12Brody M.S. Vijay K. Price C.W. J. Bacteriol. 2001; 183: 6422-6428Crossref PubMed Scopus (79) Google Scholar), whereas environmental stresses such as heat or salt shock, or ethanol treatment, activate RsbU, via the kinase RsbT (7Yang X. Kang C.M. Brody M.S. Price C.W. Genes Dev. 1996; 10: 2265-2275Crossref PubMed Scopus (236) Google Scholar, 13Kang C.M. Brody M.S. Akbar S. Yang X. Price C.W. J. Bacteriol. 1996; 178: 3846-3853Crossref PubMed Google Scholar, 14Völker U. Dufour A. Haldenwang W.G. J. Bacteriol. 1995; 177: 114-122Crossref PubMed Google Scholar). Dephosphorylated RsbV subsequently liberates σB from the transcriptionally inactive σB·RsbW complex, by competing with σB for binding surfaces on RsbW (15Alper S. Dufour A. Garsin D.A. Duncan L. Losick R. J. Mol. Biol. 1996; 260: 165-177Crossref PubMed Scopus (107) Google Scholar, 16Delumeau O. Lewis R.J. Yudkin M.D. J. Bacteriol. 2002; 184: 5583-5589Crossref PubMed Scopus (59) Google Scholar), freeing σB to bind to core RNA polymerase to activate transcription of the σB regulon. The alternative binding of RsbW to σB or RsbV is a regulatory mechanism called "partner switching" (17Alper S. Duncan L. Losick R. Cell. 1994; 77: 195-205Abstract Full Text PDF PubMed Scopus (185) Google Scholar).RsbT is related to the anti-σ factors RsbW and SpoIIAB; all are members of the GHKL family of kinases/ATPases, a group that includes two-component histidine kinases, topoisomerases, and chaperones. However, RsbT does not bind, as in the other partner-switching mechanisms, to σ or anti-anti-σ factors. During exponential growth RsbT is thought to be sequestered in a large supramolecular complex composed of RsbR and RsbS (18Chen C.-C. Lewis R.J. Harris R. Yudkin M.D. Delumeau O. Mol. Microbiol. 2003; 49: 1657-1669Crossref PubMed Scopus (88) Google Scholar). However, during environmental stress RsbT is liberated from the supramolecular complex after phosphorylating its substrates, RsbR and RsbS. RsbT is then free to associate with, and activate, RsbU (7Yang X. Kang C.M. Brody M.S. Price C.W. Genes Dev. 1996; 10: 2265-2275Crossref PubMed Scopus (236) Google Scholar). RsbU is not a substrate for the kinase activity of RsbT (19Kang C.M. Vijay K. Price C.W. Mol. Microbiol. 1998; 30: 189-196Crossref PubMed Scopus (53) Google Scholar), and the precise mechanism of the activation of RsbU by RsbT remains unknown. Analysis of the sequence of RsbU shows that it is composed of two domains, a C-terminal domain of ∼200 amino acids with sequence homology to other PP2C-type phosphatases and a N-terminal domain of ∼110 amino acids with no significant homology to any non-RsbU sequences. The simplest hypothesis regarding the role of the N-terminal domain of RsbU is that it exerts an inhibitory influence on the C-terminal, catalytic domain, which is relieved by the binding of RsbT to RsbU.We report here an investigation into the activation process of RsbU that integrates genetics and molecular and structural biology. The phenotype of B. subtilis strains containing deletions in the N-terminal domain of RsbU support the view that this domain plays a critical role in the activation of the phosphatase by RsbT. These results are discussed in the light of our determination of the crystal structure at 1.6-Å resolution of the N-terminal domain of RsbU, and conclusions are drawn as to the nature of the activation process of RsbU.EXPERIMENTAL PROCEDURESBacterial Strains, Media, and Growth Conditions—The experiments conducted in this study (Table I) were performed with derivatives of B. subtilis wild type strain 168 (20Kunst F. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3102) Google Scholar). For the construction of the various truncated versions of RsbU, there were two approaches. First, the rsbU gene, and truncated versions thereof, were amplified by PCR with chromosomal DNA of B. subtilis strain 168 as the template using appropriate primer pairs and a proofreading Taq polymerase. Subsequently, all these PCR products were digested with HindIII and SalI and ligated into the non-integrative plasmid pDG148 (21Stragier P. Bonamy C. Karmazyn-Campelli C. Cell. 1988; 52: 697-704Abstract Full Text PDF PubMed Scopus (302) Google Scholar), which had also been digested with the same enzymes. The different rsbU alleles were thus placed under the control of the IPTG-regulated promoter Pspac. After transformation into Escherichia coli strains TOP10 or TG2, and confirmation of the correct DNA sequence of the rsbU inserts by sequencing, plasmids carrying the wild type rsbU sequence (pGK01) and the truncations ΔrsbU1–19 (pMB21), ΔrsbU1–38 (pGK02), ΔrsbU1–77 (pGK03), ΔrsbU1–93 (pGK04), and ΔrsbU1–134 (pGK05) were selected for transformation into B. subtilis BSA140 (Table I). The B. subtilis strain BSA140 carries a ctc::lacZ transcriptional reporter gene fusion, but lacks a functional copy of rsbU because of a deletion of an NdeI fragment internal to the rsbU structural gene. Transformants were selected for their resistance to kanamycin (20 μg ml-1) creating strains BSG14, BSG15, BSG16, BSG17, BSG18, and BSG19, respectively (Table I).Table IPlasmids and strains used in this studyStrain or plasmidRelevant genotype or features of plasmidConstruction or Ref.B. subtilisBSA46PY22 SPβ ctc::lacZ erm cat 86aThe cat86 gene conferring chloramphenicol resistance is linked to the ctc::lacZ reporter gene fusion58Benson A.K. Haldenwang W.G. J. Bacteriol. 1992; 174: 749-757Crossref PubMed Google ScholarBSA140PY22 rsbUΔNdeI rsbX::pWH25 SPβ ctc::lacZ erm cat 86aThe cat86 gene conferring chloramphenicol resistance is linked to the ctc::lacZ reporter gene fusionspcbThe spc gene conferring resistance to spectinomycin was introduced by a Campbell-like recombination into the B. subtilis chromosome and is linked to the rsbUΔNdeI allele14Völker U. Dufour A. Haldenwang W.G. J. Bacteriol. 1995; 177: 114-122Crossref PubMed Google ScholarBSG14PY22 rsbUΔNdeI rsbX::pWH25 pGK01 SPβ ctc::lacZ erm cat 86aThe cat86 gene conferring chloramphenicol resistance is linked to the ctc::lacZ reporter gene fusionspcbThe spc gene conferring resistance to spectinomycin was introduced by a Campbell-like recombination into the B. subtilis chromosome and is linked to the rsbUΔNdeI alleleThis studyBSG15PY22 rsbUΔNdeI rsbX::pWH25 pGK02 SPβ ctc::lacZ erm cat 86aThe cat86 gene conferring chloramphenicol resistance is linked to the ctc::lacZ reporter gene fusionspcbThe spc gene conferring resistance to spectinomycin was introduced by a Campbell-like recombination into the B. subtilis chromosome and is linked to the rsbUΔNdeI alleleThis studyBSG16PY22 rsbUΔNdeI rsbX::pWH25 pGK03 SPβ ctc::lacZ erm cat 86aThe cat86 gene conferring chloramphenicol resistance is linked to the ctc::lacZ reporter gene fusionspcbThe spc gene conferring resistance to spectinomycin was introduced by a Campbell-like recombination into the B. subtilis chromosome and is linked to the rsbUΔNdeI alleleThis studyBSG17PY22 rsbUΔNdeI rsbX::pWH25 pGK04 SPβ ctc::lacZ erm cat 86aThe cat86 gene conferring chloramphenicol resistance is linked to the ctc::lacZ reporter gene fusionspcbThe spc gene conferring resistance to spectinomycin was introduced by a Campbell-like recombination into the B. subtilis chromosome and is linked to the rsbUΔNdeI alleleThis studyBSG18PY22 rsbUΔNdeI rsbX::pWH25 pGK05 SPβ ctc::lacZ erm cat 86aThe cat86 gene conferring chloramphenicol resistance is linked to the ctc::lacZ reporter gene fusionspcbThe spc gene conferring resistance to spectinomycin was introduced by a Campbell-like recombination into the B. subtilis chromosome and is linked to the rsbUΔNdeI alleleThis studyBSG19PY22 rsbUΔNdeI rsbX::pWH25 pMB21 SPβ ctc::lacZ erm cat 86aThe cat86 gene conferring chloramphenicol resistance is linked to the ctc::lacZ reporter gene fusionspcbThe spc gene conferring resistance to spectinomycin was introduced by a Campbell-like recombination into the B. subtilis chromosome and is linked to the rsbUΔNdeI alleleThis studyPB291PB2 ΔrsbU::ermC SPβctc-lacZ trpC224Wise A.A. Price C.W. J. Bacteriol. 1995; 177: 123-133Crossref PubMed Google ScholarE. coliBL21(DE3)F-ompT hsdSB(rB-mB-) gal dcm (DE3)NovagenB834(DE3)F-ompT hsdSB(rB-mB-) gal dcm met (DE3)NovagenTOP10F-mcrA Δ (mrr-hsdRMS-mcrBC) φ80lacZΔM15ΔlacX74recA1 deoR araΔ139 Δ (ara-leu)7697 galU galK rpsL (StrR) endA1 nupGInvitrogenTG2F-traD36 lacIq Δ (lacZ)M15 proA+B+/ supE hsdΔ5 thi Δ (lac-pro) Δ (srl-recA)306::Tn10 (TetR)Bethesda Research Laboratories, USAPlasmidsPAW70bla cat amyE::(PA-rsbR-rsbS-rsbT-rsbU)24Wise A.A. Price C.W. J. Bacteriol. 1995; 177: 123-133Crossref PubMed Google ScholarPMLKbla cat amyE::23Karow M.L. Piggot P.J. Gene (Amst.). 1995; 163: 69-74Crossref PubMed Scopus (63) Google ScholarpET15bamp lacI PT7lacNovagenPETNRsbUBamp lacI PT7lac::rsbU1-11226Dutta S. Lewis R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 191-193Crossref PubMed Scopus (5) Google ScholarPETNRsbULamp lacI PT7lac::rsbU1-84This studyPETCRsbUamp lacI PT7lac::rsbU118-33526Dutta S. Lewis R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 191-193Crossref PubMed Scopus (5) Google ScholarpDG148bla kan pleo lacI Pspac21Stragier P. Bonamy C. Karmazyn-Campelli C. Cell. 1988; 52: 697-704Abstract Full Text PDF PubMed Scopus (302) Google ScholarpGK01bla kan pleo lacI Pspac::rsbUThis studypGK02bla kan pleo lacI Pspac::ΔrsbU1-38This studypGK03bla kan pleo lacI Pspac::ΔrsbU1-77This studypGK04bla kan pleo lacI Pspac::ΔrsbU1-93This studypGK05bla kan pleo lacI Pspac::ΔrsbU1-134This studypMB21bla kan pleo lacI Pspac::ΔrsbU1-19This studya The cat86 gene conferring chloramphenicol resistance is linked to the ctc::lacZ reporter gene fusionb The spc gene conferring resistance to spectinomycin was introduced by a Campbell-like recombination into the B. subtilis chromosome and is linked to the rsbUΔNdeI allele Open table in a new tab Bacteria were routinely grown under vigorous agitation in a minimal medium described previously (22Stülke J. Hanschke R. Hecker M. J. Gen. Microbiol. 1993; 139: 2041-2045Crossref PubMed Scopus (201) Google Scholar) supplemented with 0.2% (w/v) glucose as a carbon source and l-tryptophan (0.78 mm). The cultures were inoculated from overnight cultures propagated in minimal media containing kanamycin to an optical density at 540 nm of 0.05. The expression of the plasmid-encoded rsbU variants was induced by the addition of IPTG to a final concentration of 1 mm. Ethanol stress was imposed on the cells during exponential growth phase (A540 = 0.3) by the addition of ethanol to a final concentration of 4% (v/v).In the second approach, B. subtilis strain PB291, previously deleted for rsbU, was transformed with derivatives of plasmid pMLK (23Karow M.L. Piggot P.J. Gene (Amst.). 1995; 163: 69-74Crossref PubMed Scopus (63) Google Scholar), which directs integration at the amyE locus. A fragment of DNA corresponding to PA-rsbR-rsbS-rsbT-rsbU (where PA represents the σA-dependent promoter region of the operon) was excised from plasmid pAW70 (24Wise A.A. Price C.W. J. Bacteriol. 1995; 177: 123-133Crossref PubMed Google Scholar) by BamHI restriction digest and cloned in pMLK using the BamHI site in amyE. For the cloning of rsbR, rsbS, rsbT, and a fragment of rsbU coding for the C-terminal catalytic domain only (ΔN-rsbU-(1–112), the regions corresponding to PA-rsbR-rsbS-rsbT and ΔN-rsbU-(1–112) were amplified separately by PCR using primers that allow overlaps between the 3′ and 5′ ends, respectively. The annealed PCR products were subsequently used as the template for a further round of PCR to yield a DNA fragment of PA-rsbR-rsbS-rsbT-ΔN-rsbU-(1–112), which was also cloned in pMLK at the BamHI site in amyE. Transformants of these strains, which are diploid for rsbR, rsbS, and rsbT, were selected by loss of amylase activity on starch plates. Bacteria were grown in buffered LB, and subjected to 4% ethanol stress at time 0.In both cases, the β-galactosidase activity of the ctc::lacZ reporter gene fusion was determined by harvesting 1-ml aliquots of cells at appropriate time points by centrifugation at 4 °C. β-Galactosidase enzyme assays were conducted as previously described (14Völker U. Dufour A. Haldenwang W.G. J. Bacteriol. 1995; 177: 114-122Crossref PubMed Google Scholar, 25Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar).Purification of Full-length RsbU and Truncated Proteins—RsbU was overexpressed in E. coli (BL21) and was purified using a procedure slightly modified from that previously published (18Chen C.-C. Lewis R.J. Harris R. Yudkin M.D. Delumeau O. Mol. Microbiol. 2003; 49: 1657-1669Crossref PubMed Scopus (88) Google Scholar). Cells were disrupted by sonication in 30 ml of lysis buffer (50 mm Tris-HCl, pH 8, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 10 mm MgCl2) and centrifuged for 30 min at 15,000 rpm. The supernatant was then applied to a DEAE-Sepharose (Amersham Biosciences) column pre-equilibrated with 50 mm Tris-HCl, pH 8, 1 mm dithiothreitol, 10 mm MgCl2 and the chromatogram was developed with a NaCl gradient from 0 to 400 mm. The RsbU-containing fractions were concentrated by centrifugal filtration (Amicon) and then loaded onto a Superdex-200 gel filtration column (Amersham Biosciences). To remove the few remaining contaminants, RsbU was further purified using high resolution Mono-Q ion exchange chromatography using the same buffer conditions as for the earlier DEAE-Sepharose column. For C-RsbU, which was cloned into pET15b, the overexpression and purification protocols were similar to those for full-length RsbU, except that a Superdex-75 gel filtration column was used instead of Superdex-200. The two N-RsbU constructs (residues 1–84 and 1–112) were purified as described previously (26Dutta S. Lewis R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 191-193Crossref PubMed Scopus (5) Google Scholar); RsbT was purified as described previously (18Chen C.-C. Lewis R.J. Harris R. Yudkin M.D. Delumeau O. Mol. Microbiol. 2003; 49: 1657-1669Crossref PubMed Scopus (88) Google Scholar) except that all buffers were supplemented with 100 μm ATP to maintain RsbT in a soluble form. Estimates of the molecular sizes of RsbU, N-RsbU, and C-RsbU were obtained by the use of Superdex-200 (Amersham Biosciences) gel filtration chromatography, calibrated against proteins of known molecular sizes.Measurement of the Phosphatase Activity—RsbV-P, the phosphatase substrate, was prepared as previously described (18Chen C.-C. Lewis R.J. Harris R. Yudkin M.D. Delumeau O. Mol. Microbiol. 2003; 49: 1657-1669Crossref PubMed Scopus (88) Google Scholar). The dephosphorylation reactions were performed in a buffer of 50 mm Tris-HCl, pH 7.5, 50 mm KCl, 10 mm MgCl2,1mm MnCl2,and1mm dithiothreitol at 30 °C with 30 μm RsbV-P, 0.5 μm RsbU (or C-RsbU) and, unless specified, 1 μm RsbT. The rates of dephosphorylation of RsbV-P were measured at various time intervals by removing 20-μl samples, stopping the reaction in each by the addition of 10 μl of loading buffer (40% glycerol, 200 mm EDTA, and 0.1% bromphenol blue) and placing the sample on ice until analysis by native gel electrophoresis and Coomassie Blue staining. RsbV-P and RsbV bands are easily separated on a 12% acrylamide gel (16Delumeau O. Lewis R.J. Yudkin M.D. J. Bacteriol. 2002; 184: 5583-5589Crossref PubMed Scopus (59) Google Scholar). Eight time points were normally taken per reaction. The gels were scanned and intensities of the bands corresponding to the appearance of RsbV were measured with Scion Image software. The values were then compared with a standard curve of known concentrations of RsbV treated under the same electrophoretic conditions.Crystallographic Methods—The structure of N-RsbU was determined by selenomethionine MAD phasing. To prepare selenomethionyl-labeled N-RsbU, the E. coli methionine auxotroph, B834 (DE3), was transformed with pETNRsbUL, a pET15b derivative that directs the IPTG-inducible expression of N-RsbU-(1–112) (26Dutta S. Lewis R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 191-193Crossref PubMed Scopus (5) Google Scholar). Transformants were initially grown in LB media, before harvesting, washing, and finally inoculating selenomethionyl media. This culture was grown for a further 4 h before the addition of IPTG to a final concentration of 1 mm and the cells were harvested by centrifugation 4 h later, and the cell pellets were stored at -80 °C overnight. Purification and crystallization were carried out as reported previously (26Dutta S. Lewis R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 191-193Crossref PubMed Scopus (5) Google Scholar). The near complete incorporation of selenomethionine was confirmed by mass spectrometry.MAD diffraction data were collected at beamline BM14 of the ESRF, Grenoble, France, from a single crystal of SeMet N-RsbU at three different wavelengths to ∼2.1-Å resolution. Each data set was individually integrated and reduced using the HKL suite (27Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38355) Google Scholar) scaling the Bijvoet pairs separately. Native diffraction data were collected separately on ID14-EH2 to a resolution of 1.6 Å for refinement purposes. Data collection statistics are summarized in Table II. The structure was determined with the program SOLVE in its automatic mode (28Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar). A total of two heavy atom sites were found corresponding to the two internal methionines, with a SOLVE Z-score of 9.1. MAD phasing from these sites produced an electron density map with a clear boundary between protein and solvent, and a mean FOM to 2.3 Å of 0.34. These phases were improved by density modification in RESOLVE (mean FOM of 0.47), producing an electron density map of sufficient quality for RESOLVE to automatically build amino acids 3–34 (helices 1–2) and 44–58 (helix 3) with the correct sequence. After initial refinement in REFMAC (29Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13776) Google Scholar), the higher resolution data became available and refinement proceeded against these data, where the Rfree flags were maintained from the MAD data. Building of the rest of the structure, mostly loops and helix 4, was performed by hand in QUANTA (Acclerys). All other calculations were performed using programs from the CCP4 suite (30P4 CC Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19703) Google Scholar). The loop between helices 3 and 4 was built in two stages, commencing only when its path could be traced unequivocally. Successive rounds of rebuilding and refinement were interspersed until refinement converged, at an Rwork of 19.6% and Rfree of 22.9%. Greater than 97% of the residues of the final model fall within the core region of the Ramachandran plot, and the overall G factor is 1.6. Statistics for the final model are presented in Table II. Atomic coordinates and structure factor amplitudes have been deposited with the RCSB, with accession code 1w53.Table IIData collection and refinement statistics Values in parentheses refer to the highest resolution shell. r.m.s.d., root mean square deviation.Data setSe-PeakSe-EdgeSe-RemoteNativeData collectionWavelength (Å)0.979220.979360.930280.933Resolution limits (Å)50-2.0850-2.0950-2.2531-1.60Number of measurements52293345712889348906Number of reflections83668310680410180Completeness (%)95.9 (84.3)96.7 (96.5)98.6 (86.9)100 (100)RsymaRsym = Σ|I - 〈I〉|/ ΣI, where 〈I〉 is the average intensity from multiple observations of symmetry related reflections (×100)10.3 (36.9)9.7 (36.2)8.9 (20.6)7.2 (16.4)I/(σ)I15.2 (3.4)13.0 (2.8)14.0 (7.7)4.4 (4.1)RefinementNumber of non-H atoms784Average B factor all atoms (Å2)20.6RworkbRwork = Σh‖Fo| - |Fc‖/ Σh|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes (%)19.8RfreecRfree was calculated with a 5% randomly-selected subset of the reflections not used in refinement (%)22.9r.m.s.d. bond distances (Å)0.019r.m.s.d. bond angles (°)1.61r.m.s.d. B factor main chain bonds (Å2)1.53r.m.s.d. B factor side chain bonds (Å2)2.92a Rsym = Σ|I - 〈I〉|/ ΣI, where 〈I〉 is the average intensity from multiple observations of symmetry related reflectionsb Rwork = Σh‖Fo| - |Fc‖/ Σh|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudesc Rfree was calculated with a 5% randomly-selected subset of the reflections not used in refinement Open table in a new tab RESULTSEffect of Deletions of the N-terminal Domain of RsbU in Vivo—If the N-terminal domain of RsbU indeed acts as an inhibitory element preventing activation of RsbU in the absence of stress, partial or complete deletion of the N-terminal part of RsbU should render the phosphatase constitutively active. To test this hypothesis experimentally, a series of N-terminal truncated RsbU variants were designed and their effect on σB activity was tested both in exponentially growing cells and in cells exposed to ethanol, a well known environmental stress stimulus. Specifically, the truncations were of the first 19, 38, 77, 93, and 134 amino acids. A wild-type copy and each of the truncated rsbU genes were cloned by PCR into the self-replicating plasmid pDG148 and thus were placed under the control of the IPTG-inducible promoter, Pspac. Plasmids were transformed into a derivative of B. subtilis wild-type strain BSA46, in which the chromosomal copy of rsbU had been inactivated by a deletion of an internal NdeI fragment in rsbU (BSA140) (14Völker U. Dufour A. Haldenwang W.G. J. Bacteriol. 1995; 177: 114-122Crossref PubMed Google Scholar). The expression of all truncated versions of RsbU on IPTG induction was verified by Western blot analysis with monoclonal antibodies directed against RsbU (data not shown). σB activity was monitored under the same conditions with a ctc-lacZ reporter gene fusion. This fusion is known to be strictly σB-dependent (31Igo M. Losick R. J. Mol. Biol. 1986; 191: 615-624Crossref PubMed Scopus (132) Google Scholar) and thus the β-galactosidase activity of those strains is directly correlated to σB activity.Surprisingly, although all the RsbU variants were expressed, none of the strains harboring the plasmid-encoded truncated rsbU alleles displayed any significant ctc-lacZ reporter gene activity during exponential growth in the presence of the inducer IPTG (data not shown). Only the strain carrying a plasmid-encoded full-length copy of rsbU displayed a modest β-galactosidase, and hence σB activity, in the presence of IPTG. Therefore, partial or complete deletion of the N-terminal domain of RsbU does not render RsbU constitutively active in vivo. Next, the ability of the truncated RsbU variants to mediate environmental stress-triggered activation of σB was tested. Cells grown in the presence or absence of the inducer IPTG were exposed to 4% ethanol, a well known strong inducer of the σB regulon (32Boylan S.A. Redfield A.R. Brody M.S. Price C.W. J. Bacteriol. 1993; 175: 7931-7937Crossref PubMed Scopus (191) Google Scholar, 33Völker U. Völker A. Maul B. Hecker M. Dufour A. Haldenwang W.G. J. Bacteriol. 1995; 177: 3771-3780Crossref PubMed Google Scholar). As expe